According to generally accepted knowledge, two classes of cell organelles, i.e. plastids and mitochondria, are derived from initially independent prokaryotes that were taken up into a predecessor of present day eukaryotic cells by separate endosymbiotic events (Gray, 1991). As a consequence, these organelles contain their own DNA, DNA transcripts in the form of messenger RNA, ribosomes, and at least some of the necessary tRNAs that are required for decoding of genetic information (Marechal-Drouard et al., 1991).
While, shortly after endosymbiotic uptake, these organelles were genetically autonomous since they contained all the elements necessary to drive prokaryotic life, this autonomy was reduced during evolution by transfer of genetic information to the cell's nucleus. Nevertheless, their genetic information is of sufficient complexity to make recent cell organelles an attractive target for gene technology. This is particularly the case with plastids, because these organelles still encode about 50% of the proteins required for their main function inside the plant cell, photosynthesis. Plastids also encode their ribosomal RNAs, the majority of their tRNAs and ribosomal proteins. In total, the number of genes in the plastome is in the range of 120 (Palmer, 1991). The vast majority of proteins that are found in plastids are, however, imported from the nuclear/cytosolic genetic compartment.
Plastids Can Be Genetically Transformed
With the development of general molecular cloning technologies, it became soon possible to genetically modify higher plants by transformation. The main emphasis in plant transformation was and still is on nuclear transformation, since the majority of genes, ca. 26.000 in the case of Arabidopsis thaliana, the complete sequence of which was recently published (The Arabidopsis Genome Initiative, 2000), is found in the cell's nucleus. Nuclear transformation was easier to achieve, since biological vectors such as Agrobacterium tumefaciens were available, which could be modified to efficiently enable nuclear transformation (Galvin, 1998). In addition, the nucleus is more directly accessible to foreign nucleic acids, while the organelles are surrounded by two envelope membranes that are, generally speaking, not permeable to macromolecules such as DNA.
A capability of transforming plastids is highly desirable since it could make use of the high gene dosage in these organelles that bears the potential of extremely high expression levels of transgenes. In addition, plastid transformation is attractive because plastid-encoded traits are not pollen transmissible; hence, potential risks of inadvertent transgene escape to wild relatives of transgenic plants are largely reduced. Other potential advantages of plastid transformation include the feasibility of simultaneous expression of multiple genes as a polycistronic unit and the elimination of positional effects and gene silencing that may result following nuclear transformation.
Methods that allow stable transformation of plastids could indeed be developed for higher plants. To date, two different methods are available, i.e. particle bombardment of tissues, in particular leaf tissues (Svab et al., 1990), and treatment of protoplasts with polyethylene glycol (PEG) in the presence of suitable transformation vectors (Koop et al., 1996). Both methods mediate the transfer of plasmid DNA across the two envelope membranes into the organelle's stroma.
Conventional plastid transformation technology is described in Heifetz, 2000 and Koop et al., 1996.
Conventional plastid transformation vectors usually need to serve at least two purposes: (1) introduction of one or more desired foreign genes to be expressed by the genetic machinery of the plastids, and (2) selection of cells containing transformed plastomes by inhibitor selection and/or by screening for a detectable phenotype. Plastid transformation vectors usually contain complete gene cassettes consisting of four operable linked elements: a promoter sequence, a 5′ untranslated region, a coding region, and a 3′ untranslated region.
These cassettes, however, do not make use of the potential to co-express several genes in an operon under the control of a single promotor.
Selection is achieved either by replacing a complete resident plastid gene by a mutant gene, which confers resistance to selection inhibitors (U.S. Pat. No. 5,451,513) or by introducing a complete expression cassette, which leads to enzymatic inactivation of an inhibitor (U.S. Pat. No. 5,877,402). These marker genes that are needed for the selection of transgenic plant cells from a vast background of untransformed cells code for antibiotic or herbicide resistance genes. Examples for plastid resistance genes are aadA conferring resistance to spectinomycin and streptomycin (Svab & Maliga, 1993), or nptII conferring resistance to kanamycin (Carrer et al., 1993). As these marker genes are stably integrated into the genome together with the genes of interest (GOI), they will stay in the homoplastomic transgenic plants although they are not required for GOI function. These remaining marker genes are a main issue of criticism of plant biotechnology as they could theoretically increase antibiotic resistance of pathogens or herbicide resistance of weeds. Construction of a selection system which does not result in a resistance gene in the transgenic plant is, therefore, highly desirable (Iamtham and Day, 2000).
In addition to the two or more gene cassettes, transformation vectors usually contain flanking regions of the insertion site, which are necessary for the stable introduction of engineered sequences into the plastome by two reciprocal homologous recombination events. To this end, chloroplast transformation vectors contain chloroplast genome sequences to serve as homologous flanks. Since the chloroplast genomes of different species differ in their sequences, species-specific transformation vectors have to be used. This requires substantial effort, when cloning transformation vectors, and is in contrast to the situation in nuclear transformation.
In all conventional transformation vectors, the selection marker is flanked by sequences homologous to plastid DNA; therefore, it is stably integrated into the plastome, although it is not needed for the desired function of the sequence(s) of interest. These remaining marker genes could theoretically spread to other organisms by giving a selective advantage. Increased antibiotic resistance in pathogens might cause problems in clinical treatment. Thus, the development of a system which results in transplastomic plants not containing any selection marker is highly desirable. A further advantage of such a system would be the possibility of re-using the same marker gene for subsequent transformations, which is at present difficult due to the limited number of efficient selection markers.
Furthermore, the copy number of any transgene stably integrated into plastome molecules can obviously never exceed the plastome copy number, thus limiting the potential transgene expression level to a certain degree. Consequently, the copy number of the transgene(s) can be further increased when located on an extra-chromosomal element.
U.S. Pat. No. 5,693,507 discloses a process for introducing a heterologous DNA into a chloroplast whereby the heterologous DNA contains operably linked control elements allowing for expression in the chloroplast. The process according to U.S. Pat. No. 5,693,507 has not provided long-term maintenance of the heterologous DNA in a plastid. Moreover, the expression of the heterologous DNA in the plastid is insufficient for practical application.
Therefore, it is an object of the invention to provide an efficient and highly versatile process of genetic transformation of plant plastids whereby genetically stable transgenic plants may be produced.
It is another object of the invention to provide a process of genetic transformation of plant plastids, which gives stably transformed plants and allows very high transgene expression levels.
It is another object of the invention to provide a process of genetic transformation of plant plastids, which allows expression of multiple genes of interest (polycistronic expression).
It is a further object of the invention to provide a novel process of genetic transformation of plant plastids, which gives transgenic plants not containing a marker gene such as an antibiotic resistance gene.
It is a further object to provide vectors capable of replicating in plant cells, preferably in plastids, whereby the replication frequency of the vector is tuneable.